Research ArticleImmunology

Essential roles for Cavβ2 and Cav1 channels in thymocyte development and T cell homeostasis

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Science Signaling  20 Oct 2015:
Vol. 8, Issue 399, pp. ra103
DOI: 10.1126/scisignal.aac7538

Channeling calcium for T cell development

Ca2+ signaling in response to stimulation of the T cell receptor (TCR) is critical to the activation of transcription factors, such as nuclear factor of activated T cells (NFAT), and the development of T cells in the thymus. Jha et al. found that mice with a T cell–specific deficiency in the β2 regulatory subunit of voltage-gated calcium (Cav) channels had fewer thymocytes and peripheral T cells than did control mice. Loss of the β2 subunit resulted in decreased Ca2+ influx in thymocytes in response to TCR stimulation, defective NFAT responses, and decreased thymocyte proliferation. Together, these data suggest that Cav channels contribute to the Ca2+ influx that is required for T cell development.

Abstract

Calcium ions (Ca2+) are important in numerous signal transduction processes, including the development and differentiation of T cells in the thymus. We report that thymocytes have multiple types of pore-forming α subunits and regulatory β subunits that constitute voltage-gated Ca2+ (Cav) channels. In mice, T cell–specific deletion of the gene encoding the β2 regulatory subunit of Cav channels (Cacnb2) reduced the abundances of the channels Cav1.2 and Cav1.3 (both of which contain pore-forming α1 subunits) and impaired T cell development, which led to a substantial decrease in the numbers of thymocytes and peripheral T cells. Similar to the effect of Cacnb2 deficiency, pharmacological blockade of pore-forming Cav1α subunits reduced the sustained Ca2+ influx in thymocytes upon stimulation of the T cell receptor, decreased the abundance of the transcription factor NFATc3, inhibited the proliferation of thymocytes in vitro, and led to lymphopenia in mice. Together, our data suggest that Cav1 channels are conduits for the sustained Ca2+ influx that is required for the development of T cells.

INTRODUCTION

Calcium ions (Ca2+) are critical in numerous signal transduction processes (1), including the development and differentiation of thymocytes in the thymus to generate T cells (26). T cell development proceeds through ordered steps of cellular differentiation, lineage commitment, and selection. Immature CD4CD8 double-negative (DN) thymocytes (subcategorized as stages DN1 to DN4) develop into CD4+CD8+ double-positive (DP) thymocytes, which then give rise to CD4+ or CD8+ single-positive (SP) T cells (7). The earliest DN thymocytes have CD44, but not CD25, on their cell surface (CD44+CD25) and are called DN1 cells. Maturation of thymocytes then proceeds through the DN2 (CD44+CD25+), DN3 (CD25+CD44), and DN4 (CD25CD44) stages. At the DN3 stage, thymocytes undergo the β-selection checkpoint. DN3 cells are tested for the successful expression of a T cell receptor β (TCRβ) polypeptide in the context of the invariant pTα subunit and CD3, which together form the pre-TCR (8). Expression of rearranged TCRβ and formation of the pre-TCR complex lead to a series of events collectively known as β-selection, resulting in allelic exclusion of the TCRβ locus, expansion, and differentiation to the DP stage (8, 9). Several studies have indicated that thymocytes that successfully pass the β-selection checkpoint receive pre-TCR–induced signals for proliferation and survival (10, 11). An increase in the intracellular concentration of cytosolic calcium ([Ca2+]i) induces cell cycle progression and survival in pre-T cells (CD25+CD44 and CD25CD44 cells) (24). Indeed, stimulation of a functional pre-TCR leads to a biphasic rise in [Ca2+]i, which activates two important transcription factors: nuclear factor κB and nuclear factor of activated T cells (NFAT). Both transcription factors are necessary for β-selection and T cell differentiation (24, 12).

One well-known mechanism of Ca2+ entry is through Ca2+ release–activated Ca2+ (CRAC) channels (13). In the CRAC pathway, the Ca2+ sensor stromal interaction molecule 1 (STIM1) responds to decreases in the concentration of Ca2+ stored in the endoplasmic reticulum (ER) by associating with the CRAC channel pore subunit ORAI1 and activating the sustained influx of extracellular Ca2+ through a process called store-operated Ca2+ entry (SOCE) (13, 14). Mice that lack CRAC components show no defect in the thymic development of conventional TCRαβ+ T cells (1520). These observations were unexpected and are suggestive of a role for CRAC-independent Ca2+ influx mechanisms during conventional TCRαβ+ T cell development in the thymus. Therefore, we asked whether voltage-gated Ca2+ (Cav) channels (21) might regulate the development of T cells in the thymus.

Cav channels are Ca2+-selective channels that mediate Ca2+ influx in response to the depolarization of excitable cells, such as myocytes, cardiomyocytes, and neurons (22, 23). These channels have been subdivided into three structurally and functionally related families: Cav1, Cav2, and Cav3. Cav1 and Cav2 channels are multisubunit complexes consisting of a pore-forming α1 subunit; a membrane-anchored, disulfide-linked complex of α2 and δ subunits; an intracellular β subunit; and, in some cases, a transmembrane γ subunit (23, 24). Because different genes encode the ion-conducting Cavα1 subunits, many different subtypes of the Cav1 family (Cav1.1, Cav1.2, Cav1.3, and Cav1.4), Cav2 family (Cav2.1, Cav2.2, and Cav2.3), and Cav3 family (Cav3.1, Cav3.2, and Cav3.3) exist (25). The β subunits are cytoplasmic proteins that bind with high affinity to the pore-forming α1 subunit of Cav1 and Cav2 channels. They are required for correct targeting Cavα1 subunits to the plasma membrane (2629) and modulation of channel kinetics (24, 30). Four genes that encode β subunits (β1 to β4) have been identified (30). We and others demonstrated that human and mouse T cells express members of the Cav1 family (Cav1.1, Cav1.2, Cav1.3, and Cav1.4) and the genes of the regulatory β3 and β4 subunits (3137), supporting the notion that there may be a distinct requirement for Cav1 channels in T cell survival, activation, and functions (31, 32, 35, 3840). A study identified a voltage-gated Na+ channel that was essential for the positive selection of CD4+ T cells (41). Thus, the prominent role of voltage-gated Ca2+ and Na+ channels in T cells is emerging as a focus of the research community (14, 4145).

Here, we report a role for the Cav1 channel components in T cell development in the thymus. We found that thymocytes expressed several Cavβ subunits and Cavα1 pore-forming subunits of Cav1 channels. We found that Cavβ2, but not other β regulatory subunits, was a critical mediator of T cell development. T cell–specific deletion of Cavβ2 led to profound defects in the development of thymocytes and reduced numbers of peripheral T cells. Cavβ2-deficient thymocytes and CD4+ T cells were prone to die spontaneously. Similar to the effects of a deficiency in Cavβ2, pharmacological inhibition of the Cav1 channels in vivo reduced the absolute numbers of thymocytes and T cells, but not bone marrow cells, in mice. In summary, we report here a previously uncharacterized role for Cavβ2 and Cav1 channels in thymocyte development and T cell homeostasis.

RESULTS

Cavβ regulatory subunit expression varies during T cell development

Cavβ regulatory subunits exhibit regulatory effects on Ca2+ channels of the Cav1 family (23). Therefore, we examined the expression patterns of Cavβ subunits in thymocytes. We designed intron-spanning polymerase chain reaction (PCR) primer pairs for each gene and obtained the expected sizes of products after analysis (Fig. 1A). Total thymocytes had mRNAs for Cacnb1 (which encodes Cavβ1), Cacnb2 (encoding Cavβ2), and Cacnb3 (encoding Cavβ3), as well as Cacnb4 (which encodes Cavβ4), albeit at a decreased abundance (Fig. 1A). Real-time reverse transcription PCR (RT-PCR) analysis of sorted thymocyte subsets from wild-type mice showed that Cacnb2 mRNA was the most abundant in DN thymocytes and that it decreased in abundance as the cells matured to become DP thymocytes (Fig. 1B). Cacnb2 mRNA abundance was then increased in CD4 SP thymocytes; however, it remained reduced in abundance in CD8 SP thymocytes (Fig. 1B). The mRNAs of the other Cavβ subunits, including Cacnb1 and Cacnb3, were low in abundance in purified DN subsets (CD4CD8CD19); instead, they were more abundant in DP thymocytes and mature thymocytes (Fig. 1B). Together, these data suggest that the Cavβ regulatory proteins might play a role in T cell development.

Fig. 1 Expression analysis of Cavβ regulatory subunits in thymocytes.

(A) Total RNA was isolated from the thymi and hearts of 6- to 8-week-old wild-type (WT) C57BL/6J mice and subjected to RT-PCR analysis of the abundances of mRNAs encoding the regulatory β subunits of Cav channels: Cavβ1 (Cacnb1), Cavβ2 (Cacnb2), Cavβ3 (Cacnb3), and Cavβ4 (Cacnb4). Hprt mRNA was used as a loading control. Data are representative of four independent experiments. (B) Thymocytes from WT mice were sorted into pure populations (>99%) of DN (CD4CD8CD19), DP (CD4+CD8+CD19), CD4 SP (CD4+CD8CD19), and CD8 SP (CD4CD8+CD19). Total RNA was isolated from these populations and subjected to real-time PCR analysis of the abundances of Cacnb1, Cacnb2, and Cacnb3 mRNAs, as indicated. GAPDH mRNA abundance was used for normalization. Data are means ± SD of five mice from four independent experiments.

Loss of Cavβ2 results in reduced numbers of thymocytes

To investigate the role of Cavβ subunits in T cell development, we analyzed thymic T cell populations in mice deficient in individual Cavβ subunits. Because the development of T cells in the thymi of mice deficient in either Cavβ3 (34) or Cavβ4 (31) is normal, we focused on Cavβ2 and analyzed mice homozygous for the floxed Cacnb2 allele (Cacnb2fl/fl) (46). Germ-line deletion of Cacnb2 causes early embryonic death (46, 47). To delete Cacnb2 specifically during the development of T cells in the thymus, we bred Cacnb2fl/fl mice (46) with mice expressing a Cre transgene under the control of the proximal promoter of lck (which encodes lymphocyte protein tyrosine kinase), which is first expressed at the DN2 to DN3 stage of thymocyte development (Lck-Cre) (48). Because there was no difference between T cells from Cacnb2+/+Lck-Cre+ and those from Cacnb2fl/fl mice, we also used littermate Cacnb2fl/fl control mice in some experiments. Hereafter, we refer to littermate control mice as Cacnb2Cre/+ mice, whereas we refer to Cacnb2fl/flLck-cre+ mice (those with a T cell–specific deletion of Cavβ2) as Cacnb2Cre/− mice. Unless otherwise stated, we used littermate controls in all experiments.

Thymocyte numbers in Cacnb2Cre/− mice were consistently 10 to 20 times less than those of Cacnb2Cre/+ mice, ranging from 4 × 106 to 10 × 106 cells per Cacnb2Cre/− mouse (Fig. 2A). We found a reduction in the percentage of DP thymocytes with a concomitant increase in the percentage of DN thymocytes in Cacnb2Cre/− mice compared to those in littermate Cacnb2Cre/+ mice (Fig. 2B). The numbers of DN, DP, CD4 SP, and CD8 SP thymocytes were substantially reduced in Cacnb2Cre/− mice compared to those in littermate Cacnb2Cre/+ mice (Fig. 2C). Next, we examined the effect of the reduced numbers of CD4 SP and CD8 SP cells in the thymus on the numbers of mature CD4+ T cells and CD8+ T cells in the peripheral lymphoid organs of Cacnb2Cre/− mice (fig. S1, A and B). The spleens and peripheral lymph nodes of Cacnb2Cre/− mice had considerably fewer mature T cells than did those of Cacnb2Cre/+ mice (fig. S1, A and B). Thus, loss of Cavβ2 appeared to block thymocyte development at the DN-to-DP transition and consequently led to a reduction in the numbers of thymocytes and peripheral T cells.

Fig. 2 Defective T cell development in Cacnb2fl/flLck-Cre+ (Cacnb2Cre/− mice.

(A) Intact thymi from 4- to 5-week-old Cacnb2Cre/+ and Cacnb2Cre/− mice were isolated and the absolute numbers of live thymocytes were counted by the trypan blue exclusion method. Data are from six mice from three independent experiments. Each symbol represents a single mouse, and horizontal lines represent means. ****P < 0.0001 by unpaired t test. (B) Flow cytometric analysis of the cell surface staining of CD4 and CD8 on thymocytes from Cacnb2Cre/+ and Cacnb2Cre/− mice. Numbers in the quadrants indicate percentages. Dot plots are representative of six mice from three independent experiments. (C) Absolute numbers of the indicated populations of thymocytes from Cacnb2Cre/+ and Cacnb2Cre/− mice. Data are from six mice from three independent experiments. Each symbol represents a single mouse, and horizontal lines represent means. ****P < 0.0001 by unpaired t test.

Cavβ2 is required for the DN3-to-DN4 transition

To further define the stage at which thymocyte development was blocked during the DN-to-DP transition in Cacnb2Cre/− mice, we analyzed DN subpopulations. Real-time PCR analysis showed that Cacnb2 mRNA abundance was greatest in DN2 thymocytes and decreased with maturation (Fig. 3A). The percentage, but not the absolute number, of DN3 cells was markedly increased in Cacnb2Cre/− mice compared to that in littermate Cacnb2Cre/+ mice (Fig. 3, B and C). In contrast, the percentage, as well as the total number, of DN4 cells in Cacnb2Cre/− mice was decreased (Fig. 3, B and C). Overall, Cacnb2Cre/− mice had a reduced number of cells in DN subpopulations because of a reduction in the number of thymocytes (Fig. 3C). These data indicate that T cell development is arrested at the DN3 stage in Cacnb2Cre/− mice and suggest that Cavβ2 may be required for the DN3-to-DN4 transition.

Fig. 3 Loss of Cavβ2 prevents the transition from DN3 to DN4 cells.

(A) DN thymocytes from 6- to 8-week-old WT C57BL/6J mice were sorted into the indicated subpopulations and then subjected to real-time PCR analysis to determine the relative abundances of Cacnb2 mRNA (which encodes Cavβ2) normalized to that of Hprt mRNA. Data are means ± SD of thymi pooled from five mice from four independent experiments. (B and C) Flow cytometric analysis of the different subpopulations of DN thymocytes from the indicated mice (B) and quantification of the total numbers of DN3 and DN4 thymocytes (C). Numbers in quadrants in (B) indicate the percentages of the DN subpopulations. Dot plots are representative of six mice from three independent experiments. Data in (C) are from six mice from three independent experiments. Horizontal lines represent means. **P < 0.01, ****P < 0.0001 by unpaired t test. (D and E) Flow cytometric analysis of the different thymocyte populations from OT-II Cacnb2Cre/+ and OT-II Cacnb2Cre/− mice (D) and quantification of the numbers of total and DP thymocytes in the indicated mice (E). Numbers in quadrants in (D) indicate the percentages of the thymic populations and are representative of four mice from two independent experiments. Data in (E) are from four mice from two independent experiments. Horizontal lines represent means. ****P < 0.0001 by unpaired t test.

As was shown earlier, the loss of Cavβ2 caused a decrease in the absolute number of mature thymocytes (Fig. 2A), and we further found that the remaining thymocytes from Cacnb2Cre/− mice showed a block at the DN3 stage (Fig. 3B). However, some DN3 cells did progress to the DN4 stage and became mature CD4+ and CD8+ T cells in the periphery (fig. S1, A and B). Therefore, we wanted to address the role of Cavβ2 in the developmental process from the DN4 stage to the mature peripheral T cell stage. Because developmental defects associated with thymocyte selection can be masked by compensatory changes in the TCR repertoire, we introduced a single transgene encoding an αβ TCR into Cacnb2Cre/− (Cacnb2fl/flLck-cre+) mice to limit such compensation. We then examined the function of Cavβ2 proteins in the positive selection of thymocytes in mice expressing the transgene encoding the major histocompatibility complex class II–restricted OT-II TCR (OT-II Cacnb2Cre/− mice). Similar to mice on a nontransgenic background (Fig. 2), mice on an OT-II TCR transgenic background had reduced numbers and percentages of total thymocytes and DP thymocytes (Fig. 3, D and E). We also observed a modest reduction in the percentage of Cavβ2-deficient DP thymocytes that proceeded to the CD4+ SP stage in the OT-II Cacnb2Cre/− mice (Fig. 3D). Together, these data are suggestive of a critical role for Cavβ2 in multiple checkpoints during the thymic selection process.

Cavβ2 is necessary for the survival of thymocytes and peripheral T cells

The reduction in the number of thymocytes in Cacnb2Cre/− mice on a polyclonal (Fig. 2A) as well as TCR transgenic background (Fig. 3, D and E) suggested that Cavβ2 was required for the development of T cells. To investigate the T cell developmental defect, we analyzed the cell surface expression of the thymocyte maturation markers CD5 and CD24 (heat-stable antigen) on different thymic T cell populations. We found that the surface amount of CD5, but not CD24, was reduced on the DP thymocytes of Cacnb2Cre /− mice compared to that on DP thymocytes from control littermate mice (fig. S2). Note that the Ca2+-dependent transcription factor NFAT is required for the expression of the gene encoding CD5 in lymphocytes (49, 50).

Previous findings, including our own, support the hypothesis that L-type calcium channel components are required for the proliferation, survival, and homeostasis of peripheral T cells and mast cells (34, 51, 52); however, whether L-type calcium channel components are also required for the proliferation, survival, or homeostasis of thymocytes is unclear. Therefore, we examined the role of the Cavβ2 in the proliferation and survival of thymocytes. We assessed cell cycle progression in carboxyfluorescein diacetate succinimidyl ester (CFSE)–labeled thymocytes 3 days after they were stimulated with anti-CD3 and anti-CD28 antibodies. Whereas many wild-type thymocytes underwent cell division, most Cacnb2-deficient thymocytes failed to divide (Fig. 4A). We obtained similar results from experiments in which we monitored the incorporation of [3H]thymidine into thymocytes stimulated with anti-CD3 and anti-CD28 antibodies (fig. S3A). In addition, the amount of interleukin-2 (IL-2) secreted by Cacnb2-deficient thymocytes after stimulation with anti-CD3 and anti-CD28 antibodies (a hallmark of T cell activation) was substantially less than that secreted by similarly stimulated control thymocytes (fig. S3B). We and others previously reported a critical role for Cav channel components in mediating the survival of naïve T cells (34, 51). To assess whether Cavβ2 played a similar prosurvival role in protecting thymocytes from undergoing activation-induced cell death (AICD), we performed staining of thymocytes by annexin V and propidium iodide (PI). This staining showed that there was a greater percentage of apoptotic cells in thymocytes from Cacnb2-deficient mice than in thymocytes from control littermate mice (Fig. 4B). Finally, we examined whether Cacnb2 deficiency altered the expression of genes that encode antiapoptotic factors and found that the abundances of mRNAs for Bcl-2 and Bcl-xL were decreased in DN3 thymocytes from Cacnb2−/− compared to those in DN3 thymocytes from control mice (Fig. 4C).

Fig. 4 Requirement for the Cavβ2 subunit for the survival of thymocytes and T cells.

(A) Thymocytes from Cacnb2Cre/+ and Cacnb2Cre/− mice were labeled with CFSE and stimulated by anti-CD3 and anti-CD28 antibodies. Seventy-two hours later, cellular proliferation was assessed by flow cytometric analysis of CFSE dilution. Unstained and unstimulated cells (orange histogram) served as a negative control. Control cells are shown in black, and knockout cells are shown in blue. Data are representative of six experiments. (B) Thymocytes isolated from Cacnb2Cre/+ and Cacnb2Cre/− mice were stained with annexin V and PI and analyzed by flow cytometry to assess cell death. Data are representative of six experiments. (C) Sorted thymocytes from Cacnb2Cre/+ and Cacnb2Cre/− mice were subjected to real-time quantitative PCR analysis of the relative abundances of Bcl2 and Bcl2l1 mRNAs, which were normalized to that of Hprt mRNA. Data are means ± SD of three experiments. (D) Total RNA isolated from sorted naïve CD4+, naïve CD8+, effector CD4+, and effector CD8+ T cells enriched from the spleens and lymph nodes (LNs) of WT mice was subjected to real-time PCR analysis of the relative abundance of Cacnb2 mRNA, normalized to that of Hprt mRNA. Data are means ± SD of four experiments. (E) CD4+ T cells isolated from the spleen and LNs of Cacnb2Cre/+ and Cacnb2Cre/− mice were stained with annexin V and PI and analyzed by flow cytometry to assess cell death. Data are representative of six experiments. (F) A mixture of CD4+ T cells from Cacnb2Cre/−CD45.1.2+ mice and Cacnb2Cre/+CD45.2+ mice (left) was transferred into Rag1−/−CD45.2+ recipient mice. Right: Four weeks later, cells recovered from the lymph nodes (LNs) and spleens of the recipient mice were analyzed by flow cytometry. Numbers inside the quadrants indicate the percentages of the recovered CD4+ T cells. (G) Splenocytes were purified from the recipient mice depicted in (E) and were stimulated with anti-CD3 and anti-CD28 antibodies (left) or with PMA and ionomycin (Ion) (right). Eight hours later, the percentages of live (PI-negative) IL-2–producing cells in the indicated populations were determined by intracellular flow cytometric analysis. Dot plots in (F) and (G) are representative of five recipient mice per group from three independent experiments. Unstimulated cells served as a negative control for setting gates.

Genetic ablation of Orai1, which encodes the ion-conducting pore of the CRAC channel, does not impair the survival, homeostasis, or proliferation of naïve CD4+ T cells (16, 19), and the molecular components of Ca2+ channels in these cells remain unclear. Because peripheral naïve CD4+ T cells (CD4+CD62LhighCD44low) also had abundant Cacnb2 mRNA (Fig. 4D), we investigated the effect of Cavβ2 deficiency on these naïve peripheral CD4+ T cells. We found that Cacnb2-deficient naïve CD4+ T cells in the lymph nodes and spleen underwent AICD (Fig. 4E) similarly to Cavβ2-deficient thymocytes (Fig. 4B). About 30 to 50% of unstimulated Cacnb2-deficient T cells were positive for annexin V and PI compared to only 10 to 23% of control cells (Fig. 4E), which suggested that the Cacnb2-deficient T cells were undergoing spontaneous apoptosis in vivo most likely as a result of a defect in homeostatic maintenance. These findings highlight the complex array of Ca2+ channels in T cells, which require the Cav1 channel machinery for their survival and maintenance (34, 51), which is in contrast to the function of the ORAI1 channel, which is required for mediating T cell apoptosis (53).

To evaluate the homeostatic maintenance of Cacnb2-deficient T cells in vivo, we crossed Cacnb2Cre/− mice with C57BL/6J CD45.1+ mice and generated several congenic combinations for mixed transfer experiments. We adoptively transferred a mixture of approximately equal numbers of Cacnb2Cre/− (CD45.1+CD45.2+) and Cacnb2Cre/+ (CD45.1CD45.2+) naïve CD4+ T cells (donor) into Rag-1−/− (CD45.1CD45.2+) recipient mice and analyzed the persistence of these cells over time. In contrast to wild-type donor cells (Cacnb2Cre/+), whose proportions increased over time, the proportion of transferred cells that were Cacnb2-deficient (Cacnb2Cre/−) decreased substantially over time (Fig. 4F). The ability of each transferred cell population to produce IL-2 upon stimulation by anti-CD3 and anti-CD28 antibodies or by PMA (phorbol 12-myristate 13-acetate) and ionomycin also differed, with a substantial reduction seen in the Cacnb2-deficient cells (Fig. 4G). These data suggest that there was an intrinsic defect in the survival, homeostasis, and IL-2 production of Cacnb2-deficient mature CD4+ T cells.

We also found that the abundance of Cacnb2 mRNA was greater in effector CD4+ T cells than in effector CD8+ T cells (Fig. 4D). Therefore, we next addressed the function of Cavβ2 in the TCR responses of effector CD4+ T cells. To circumvent the obstacle that mature CD4+ T cells cannot develop in the absence of Cavβ2 because of a survival defect, we deleted Cacnb2 in activated T cells from Cacnb2fl/fl mice by retrovirally expressing the enzyme Cre in these cells. We used green fluorescent protein (GFP)–tagged bicistronic retroviral vectors, which enabled us to distinguish retrovirally transduced (GFP+) cells from nontransduced (GFP) cells. As a control, we used CD4+ T cells infected with retrovirus expressing GFP and luciferase (Luc) in parallel. After Cre-expressing effector CD4+ T cells were restimulated through the TCR or by PMA and ionomycin, Cre-GFP+–expressing cells and control Luc-GFP+ cells produced similar amounts of the cytokine interferon-γ (IFN-γ) (fig. S4A). CD4+ T cells transduced with control virus did not show any difference in IFN-γ production regardless of GFP expression (fig. S4A), suggesting that the overexpression of GFP and luciferase did not impair the ability of effector CD4+ T cells to produce IFN-γ. The percentage of Cre-GFP+–expressing cells was markedly reduced compared to the percentage of control Luc-GFP+ cells, which suggests that Cacnb2-deficient effector T cells may have undergone increased cell death (fig. S4A). To directly examine whether Cavβ2 played a role in the survival of effector CD4+ T cells, we transduced Cacnb2fl/fl T cells with a retrovirus expressing Cre-GFP and analyzed apoptosis by labeling the cells with PI. We found that there was a substantial increase in the percentage of PI-positive cells among the GFP+ population (which were Cacnb2-deficient because of the expression of Cre) compared to that among the GFP-negative cells (in which Cacnb2 expression was intact) under the same culture conditions (fig. S4B). Consistent with this, we observed a defect in the survival and homeostasis of Cacnb2-deficient effector CD4+ T cells in vivo (Fig. 4F). Together, these data suggest that Cavβ2 may have a role in the survival of peripheral T cells that is distinct from its role during T cell selection in the thymus.

Cav1 channel activity is critical for T cells in the thymus and the periphery

The Cavβ subunits play an essential role in the trafficking of the pore-forming α1 subunit of Cav1 channels to the plasma membrane. In the absence of Cavβ, the cell surface abundance of Cav1 channels is low, and only very small, if any, currents can be recorded (54). Therefore, we hypothesized that thymocytes deficient in Cavβ2 might lack the cell surface expression of the Cavα1 pore-forming subunit. First, we assessed which Cavα subunits were expressed in wild-type thymocytes. RT-PCR analysis with intron-spanning primers showed that thymocytes expressed mRNAs for Cacna1c (encoding Cav1.2) and Cacna1d (encoding Cav1.3), Cacna1s (encoding Cav1.1), and Cacna1f (encoding Cav1.4) (Fig. 5A). We also detected mRNAs for Cacna1a (encoding Cav2.1), Cacna1b (encoding Cav2.2), and Cacna1e (encoding Cav2.3) in thymocytes (Fig. 5A). Through Western blotting analysis, we detected the loss of pore-forming α subunits of Cav1.2 and Cav1.3 channels in Cavβ2 -deficient thymocytes (Fig. 5B), which suggests that Cavβ2 is required for the synthesis of Cav1.2 and Cav1.3 channels in thymocytes.

Fig. 5 Expression and function of Cavα pore-forming subunits in thymocytes.

(A) Total RNA isolated from the thymi and hearts of 6- to 8-week-old WT C57BL/6J mice was subjected to RT-PCR analysis of the relative abundances of mRNAs encoding the regulatory α subunits of the Cav channels Cav1.1 (Cacna1s), Cav1.2 (Cacna1c), Cav1.3 (Cacna1d), Cav1.4 (Cacna1f), Cav2.1 (Cacna1a), Cav2.2 (Cacna1b), and Cav2.3 (Cacna1e) normalized to that of Hprt mRNA. Data are representative of four independent experiments from the same mice that were analyzed in Fig. 1A. (B) Left: Whole-cell lysates of thymocytes isolated from Cacnb2Cre/− and Cacnb2Cre/+ littermate mice were subjected to Western blotting analysis with antibodies against the indicated proteins. Western blots are representative of three independent experiments. Right: Densitometric analysis of the mean densities of bands for the indicated proteins normalized to that of actin. Data are means ± SD of three independent experiments. ***P < 0.001 by unpaired t test; ns, not significant. (C) Top: Forward-scatter (FSC) and side-scatter (SSC) patterns of CFSE-labeled WT thymocytes that were stimulated by plate-bound anti-CD3 (10 μg/ml) and soluble anti-CD28 (2 μg/ml) antibodies and then treated with vehicle control [dimethyl sulfoxide (DMSO)], the Cav1 blocker nicardipine (1 μM), the Cav2.1 blocker ω-agatoxin (1 μM), the Cav2.2 blocker conotoxin (1 μM), or the Cav2.3 blocker SNX482 (1 μM) for 72 hours. Bottom: Proliferation of thymocytes, treated as described for the top plots, was assessed after 72 hours by flow cytometric analysis of CFSE dilution. Data are representative of six independent experiments. (D) Quantification of the numbers of thymocytes, lymph node (LN) cells, splenocytes, and bone marrow cells in cohorts of 6- to 10-week-old mice that were injected intraperitoneally daily with either phosphate-buffered saline (PBS) (Control) or pharmaceutical-grade nicardipine (1 mg per mouse) for 3 weeks. Data are from five mice per group from two independent experiments. Each symbol represents a single mouse, and horizontal lines represent means. ***P < 0.001 by unpaired t test; ns, not significant.

These results prompted us to examine the population of T cells in the thymi of Cavα1-deficient mice. We stained thymocytes isolated from fetal liver–transplanted Cacna1s mutant Rag1−/− mice (Cav1.1−/− thymocytes), from Cacna1cfl/fl mice (55) crossed with mice expressing a Cre transgene under the control of the lck promoter (Cav1.2−/− thymocytes), and from Cacna1d-deficient mice (Cav1.3−/− thymocytes) (56). We also used littermate controls for each genotype. We found that the relative percentages of all thymocyte populations from these mice were normal (fig. S5), which indicated that T cell development was unaffected by the loss of individual Cav1 channels. These data suggested that individual Cavα1 calcium channels might play compensatory roles in the regulation of T cell development.

To circumvent these compensatory mechanisms, we took advantage of the widely used and clinically established dihydropyridine (DHP)–type Ca2+ channel blocker nicardipine to effectively inhibit all four DHP-sensitive Cavα1 channels (Cav1.1 to Cav1.4) (57). A low concentration of nicardipine blocked the proliferation of thymocytes upon TCR activation (Fig. 5C). Because thymocytes also expressed transcripts of Cav2 family members, we also investigated the roles of these channels in thymocyte proliferation. Blockade of Cav2.1 (with ω-agatoxin), Cav2.2 (with conotoxin), or Cav2.3 (with SNX482) did not impair the ability of thymocytes to proliferate upon TCR stimulation (Fig. 5C). This finding suggests that Cav1, but not Cav2, channels are critical for T cell proliferation in the thymus. Nicardipine inhibited the TCR-stimulated production of IL-2 by thymocytes in a dose-dependent manner and completely blocked IL-2 production when used at 1 μM (fig. S6A). The same concentration of nicardipine did not inhibit the TCR-stimulated production of IL-2 by peripheral CD4+ T cells (fig. S6B). Indeed, a higher concentration of nicardipine (>5 μM) was required to effectively inhibit IL-2 production by these cells (fig. S6B), which suggests that peripheral CD4+ T cells are less sensitive to the effects of nicardipine than are thymocytes. To further determine whether Cav1 channels were functional in human T cells, we examined the effect of nicardipine on IL-2 production by monocyte-depleted, human peripheral blood mononuclear cells (PBMCs) in vitro. Similarly to its effects on mouse T cells, nicardipine inhibited IL-2 production by human PBMCs in response to TCR stimulation (fig. S6C).

To investigate the effect of nicardipine on the mouse immune system, cohorts of young wild-type C57BL/6 mice were injected intraperitoneally daily with nicardipine or vehicle control at a similar dose, which was reported to inhibit the inflammatory response in a mouse model of asthma (58), and we analyzed the immune systems of the mice after 3 weeks. Consistent with our in vitro experiments, we found that nicardipine resulted in reduced cell numbers only in the thymus and peripheral lymphoid organs (Fig. 5D). In addition, nicardipine did not affect cell numbers in the bone marrow (Fig. 5D). Together, these data suggest that functional Cav1 channels are required for the proliferation of thymic and peripheral T cells.

The Ca2+-NFAT pathway in thymocytes is dependent on Cav1 channels

We know that the pre-TCR–mediated survival and transition of early T cells in the thymus require sustained Ca2+ entry (4); however, how this Ca2+ flux is mediated is unclear. Our earlier observation of the requirement for T cells to express Cav1 channels during thymocyte development suggests that this component may be critical for Ca2+ flux. To determine whether Cav1 channels regulated Ca2+ entry, we stimulated the TCRs of total thymocytes by cross-linking and then monitored changes in the concentration of cytosolic Ca2+ with the Ca2+-sensitive, cell-permeable fluorescent dye Fura-2 AM. TCR stimulation leads to a biphasic rise in the intracellular Ca2+ concentration in the cytosol. The initial response is believed to result from Ca2+ release from the ER (1). The decrease in the amount of Ca2+ stored in the ER stimulates sustained Ca2+ influx from outside the cell (59, 60), which is necessary to activate the NFATs, transcription factors that are critical for the development and functions of T cells (5, 6, 61). To investigate whether inhibition of Cav1 channels by nicardipine affected the cytosolic Ca2+ response in thymocytes, we stimulated wild-type thymocytes in the presence of nicardipine or vehicle control and measured [Ca2+]i with a ratiometric method. Nicardipine-treated thymocytes showed reduced Ca2+ influx in response to stimulation with an anti-CD3 antibody or to passive depletion of ER Ca2+ stores with thapsigargin (Fig. 6, A and B). These data suggest that Cav1 channels contribute to the sustained Ca2+ entry in thymocytes.

Fig. 6 Cav1 channel activity contributes to Ca2+ signaling and Nfatc3 expression in thymocytes.

(A) Thymocytes (1 × 106 cells) from WT mice were pretreated with vehicle (black lines) or nicardipine (blue lines) before being analyzed for intracellular Ca2+ mobilization in response to antibody-mediated cross-linking of the TCR and then treatment with ionomycin (Ion) in the presence of 2 mM extracellular Ca2+. (B) Thymocytes (1 × 106 cells) from WT mice were pretreated with vehicle (black lines) or nicardipine (blue lines) before being analyzed for intracellular Ca2+ mobilization in response to 1 μM thapsigargin (TG) in the absence or presence of 2 mM extracellular Ca2+. Data in (A) and (B) are representative of three independent experiments. (C) Left: WT thymocytes were treated overnight with DMSO or 1 μM nicardipine before being subjected to Western blotting analysis with antibodies against the indicated proteins. Western blots are representative of three experiments. Right: Densitometric analysis of the mean densities of NFATc3 bands normalized to those of actin bands. Data are means ± SD of three independent experiments. *P < 0.05 by unpaired t test. (D) Left: Whole-cell lysates of thymocytes isolated from Cacnb2Cre/− and Cacnb2Cre/+ littermate mice were analyzed by Western blotting with antibodies against the indicated proteins. Western blots are representative of three experiments. Right: Densitometric analysis of the mean densities of NFATc3 bands normalized to those of actin bands. Data are means ± SD of three independent experiments. *P < 0.05 by unpaired t test. (E) Total RNA isolated from sorted CD4+ T cells from Cacnb2Cre/−CD45.1.2+ or Cacnb2Cre/+CD45.2+ cells that were adoptively transferred to five mice were subjected to real-time PCR analysis to determine the abundances of nfatc3 (left) and nfatc1 (right) mRNAs, normalized to that of Hprt mRNA. Data are means ± SD of three independent experiments.

The role of Ca2+-NFAT signaling in T cell development, as well as in peripheral T cell activation, has been intensely studied. Among four members of the NFAT family, NFATc3 is the most abundant in thymocytes, and its specific deletion in these cells causes a partial block at the DN3 stage, as well as a partial block in positive selection (6). To examine the effect of the blockade of Cav1 channels on NFATc3 synthesis, we monitored the abundance of NFATc3 protein in thymocytes treated with DMSO or nicardipine. Blockade of Cav1 channels for 12 hours led to a substantial decrease in the abundance of NFATc3, but not that of another survival factor, AKT (Fig. 6C). To investigate whether the lower abundance of NFATc3 protein was a result of altered gene expression, we used quantitative RT-PCR analysis to measure the abundance of Nfatc3 mRNA in thymocytes treated with vehicle control or nicardipine for 12 hours. We found that Nfatc3 mRNA abundance was reduced in nicardipine-treated thymocytes compared to that in DMSO-treated thymocytes (fig. S7). To examine the effect of Cacnb2 deficiency on NFATc3 protein abundance in thymocytes, we performed Western blotting analysis of whole-cell lysates of thymocytes from Cacnb2Cre/+ and Cacnb2Cre/− mice. We found that NFATc3 abundance was reduced in Cacnb2Cre/− thymocytes compared to that in Cacnb2Cre/+ thymocytes (Fig. 6D). Previously, we demonstrated that Cavβ2-dependent Ca2+ entry is required for the steady-state transcriptional regulation of nfatc1 in naïve CD8+ T cells, and that a deficiency in Cacnb3 alters naïve CD8+ T cell homeostasis (34). To investigate whether nfatc1 or nfatc3 mRNA abundances were affected by the loss of Cacnb2 in peripheral CD4+ T cells, we isolated RNA from sorted CD4+ T cells from Cacnb2Cre/−CD45.1.2+ or Cacnb2Cre/+CD45.2.2+ cells from adoptively transferred mice (Fig. 4F) and subjected them to real-time PCR analysis. We found that the abundances of nfatc1 and nfatc3 mRNAs were reduced in Cacnb2Cre/− CD4+ T cells compared to those in Cacnb2Cre/+ cells. Together, these data suggest that Cav1-dependent Ca2+ entry is required for the expression and function of different NFATs in thymocytes and peripheral T cells.

DISCUSSION

Despite the established role of the sustained influx of extracellular Ca2+ into T cells during their development in the thymus, the identity and number of plasma membrane channels that mediate sustained Ca2+ entry into developing thymocytes are unclear. A well-characterized mode of Ca2+ entry into mature T cells in peripheral lymphoid organs such as the spleen and lymph nodes is SOCE (16, 17, 19). Unexpectedly, the elimination of SOCE in stimulated DN and DP thymocytes from Stim1fl/flStim2fl/flVav-iCre mice (20), Stim1-deficient mice (17), or Orai1-deficient mice (16, 19) did not impair the development of conventional T cells. Thus, these studies hint that greater complexity is involved in Ca2+ regulation, which dynamically changes with T cell development and differentiation, and suggest that differential responses are important for functional outcomes during T cell development.

Here, we examined the expression and function of Cav1 channels in thymocytes. We showed that all four Cavβ regulatory subunits (Cavβ1 to Cavβ4) are found in thymocytes; however, T cells develop normally in the thymus in the absence the Cavβ3 (31) or Cavβ4 subunits (31). We found that the lack of Cavβ2 resulted in defective T cell development in the thymus. Because DN thymocytes accumulated at the DN3 stage, at which they receive pre-TCR signals, we conclude that Cavβ2-deficient thymocytes did not properly develop after pre-TCR signaling, which resulted in a relatively smaller population of DN4 thymocytes and reduced total thymic cellularity compared to control thymocytes expressing Cavβ2. Consequently, the lack of Cavβ2 led to substantially reduced numbers of peripheral T cells, which became even more prominent in a competitive environment in which Cavβ2-deficient T lymphocytes failed to compete with Cavβ2-expressing T lymphocytes for growth and survival factors. These data emphasize the crucial function of Cavβ2 in maintaining the peripheral T cell pool. We suggest that the effect of Cavβ2 deficiency on thymocyte survival may be a result of the reduced basal abundance of NFATc3 in Cacnb2/ thymocytes.

How does loss of Cavβ2 affect these processes in T cells? We know that the regulatory Cavβ subunit binds to a conserved domain [the α-interaction domain (AID)] of the pore-forming subunit of Cav1 channels to promote cell surface trafficking and modulate channel-gating properties (23). Our findings that Cavβ2 is critical for T cell development, survival, and homeostasis suggest that Cav1 channels control sustained Ca2+ influx into thymocytes and regulate their proliferation. A deficiency in one of the Cav1 channels, Cav1.4, results in a modest decrease in the number of mature CD4 SP thymocytes in mice, whereas the number of CD8 SP thymocytes is largely unchanged (51). Our analysis of mice deficient in the Cav1.1, Cav1.2, or Cav1.3 channels showed that there was no effect on T cell development. This result suggests that multiple Cav1 channels play a role in the regulation of T cell development.

Indeed, in the presence of the Cav1 channel blocker nicardipine, sustained Ca2+ influx was attenuated, and consequently, the proliferation of thymocytes and peripheral T cells, but not bone marrow cells, was inhibited in vitro and in vivo. The half-maximal inhibitory concentration (IC50) of nicardipine for IL-2 production by mouse CD4+ T cells (~5 μM) is different from that for IL-2 production by thymocytes (~1 μM; fig. S6A). This difference may be because of a difference in the membrane potentials of thymic and peripheral T cells or the lower sensitivity of CD4+ T cells to nicardipine. The inhibition of Cav1 channels by DHPs such as nicardipine is sensitive to membrane potential, which explains why the IC50 of nicardipine for vascular smooth muscle is substantially less than its IC50 for cardiac muscle (62). Alternatively, the low sensitivity of peripheral CD4+ T cells to nicardipine may also be caused by the expression of different splice variants of Cav1 channels between thymocytes and peripheral T cells. In peripheral T cells, several splice variants of Cav1 channels in which the voltage sensor domain and part of the DHP-binding site and EF-hand Ca2+-binding motif have been deleted were reported (32, 63). Whereas removal of the voltage sensor may alter the voltage-gated activation of this channel, partial deletion of the DHP-binding site may decrease the sensitivity of T cell–specific Cav1 channels, which may explain the large concentrations of nicardipine that are needed to completely block Ca2+ influx through Cav1 channels in peripheral T cells (64). Given the emerging importance of voltage-dependent ion channels in T cell biology (41, 44, 45, 64), it remains to be determined how Cav1 channels, which are activated by depolarization of the membrane potential, regulate the sustained Ca2+ influx that is required for the normal T cell development.

Cav1.4 channels are required for the survival and function of naïve T cells (51). In that study, currents were recorded in wild-type naïve (CD44low) mouse CD4+ and CD8+ lymphocytes, but they were absent in cells from Cacnalf−/− mice, which do not express CaV1.4 on the plasma membrane as determined by surface biotinylation experiments (51). As outlined by Niemeyer and Hoth (65), the currents measured in that study (51) were small, did not show the pronounced Ca2+-dependent inactivation (a hallmark of Cav1.4) in contrast to most Cav channels, and resulted in a current-voltage relationship that had a peak potential of more than 0 mV. It remains an intriguing finding in light of the lack of any depolarizing signal in T cells.

Our data indicate that genetic deletion of Cav1.1, Cav1.2, or Cav1.3 does not cause a defect in thymocyte development. A minor reduction in the number of CD4 SP thymocytes was observed in Cav1.4-deficient animals, which is suggestive of a potential role for Cav1.4 in the transition of DP thymocytes into CD4 SP thymocytes (51). These results led us to hypothesize that more than one Cav1 channel might play an important role during T cell development and may compensate for the lack of an individual Cav1 channel. This hypothesis was supported by the marked effect that Cavβ2 deficiency had on T cell development, which was likely a result of the lack of Cav1.2 and Cav1.3 channels in Cavβ2-deficient thymocytes. A double deficiency in Cav1.2 and Cav1.3 channels may likely block early T cell development similarly to the deficiency in Cavβ2. Whereas these observations remain to be validated in mice lacking both Cav1.2 and Cav1.3 channels (Cav1.2fl/fllck-Cre × Cav1.3−/−), the present study describes the involvement of Cavβ2 and Cav1 channels in the regulation of T cell development.

MATERIALS AND METHODS

Mice and adoptive transfer experiments

All experiments with mice were approved by the Institutional Animal Care and Use Committee of the Trudeau Institute. Cacnb2fl/+ mice were bred with Lck-Cre transgenic mice (Taconic), and the offspring were bred with Cacnb2fl/fl mice. Cacnb2Cre/+ mice were bred with congenic CD45.1 mice (available at the Trudeau Institute). We used 4- to 10-week-old mice in all experiments. All experiments used mice of mixed background (two generations backcrossed with C57BL/6 mice). For the adoptive transfer of naïve CD4+ T cells, sorted cells from Cacnb2Cre/−CD45.1.2+ mice were mixed with Cacnb2Cre/+ CD45.2+ cells in equal numbers (1:1 ratio) and then were transferred into Rag1/ CD45.2+ mice. The recipient mice were sacrificed 4 weeks later, and donor cells were detected by flow cytometric analysis of congenic markers and were gated on CD4+ T cells.

Fetal liver transfers

For transplantation experiments, embryos were generated from crosses of Cacna1smdg heterozygous mice (66), which were provided by K. Beam. Fetal livers were harvested from day 12 embryos and placed in 1 ml of Iscove’s modified Dulbecco’s medium, 2% fetal calf serum, and single-cell suspensions were prepared by passage through a 26-gauge needle. To determine genotypes rapidly, 1/20 of the fetal liver suspension was washed with 1 ml of PBS without serum, resuspended in 1× PCR buffer containing proteinase K (10 μg/ml), 0.045% NP-40, and 0.045% Tween 20, and incubated at 55°C for 30 min. The protease was inactivated by incubating the samples for 10 min at 100°C, and 2 μl of the lysate was subjected to genotyping by standard PCR analysis. Within 6 hours, fetal liver cells in 300 μl of medium were injected into the tail vein of a Rag1/ female recipient mouse that had previously been irradiated at 550 rads with a 137Cs source. Recipient mice were maintained in autoclaved cages on autoclaved water containing trimethoprim-sulfamethoxazole.

T cell purification, differentiation, and stimulation

Lymphocytes were isolated from the spleens and lymph nodes of 4- to 10-week-old mice. Peripheral T cells were purified with anti-CD4– or anti-CD8–coupled beads (Miltenyi Biotec). Unless stated otherwise in the legends, all experiments used CD4+ or CD8+ T cells that were enriched with MACS (magnetic-activated cell sorting) columns. Thymocytes were sorted into different T cell populations (gated as CD4 SP, CD8 SP, or DP) with a BD Influx cell sorter (Becton Dickinson). The isolation, culture, and differentiation of T cells and the assessment of cytokine production by intracellular staining and flow cytometric analysis were performed as described previously (34).

RNA isolation, semiquantitative RT-PCR, and real-time PCR analysis

Total RNA was isolated from cells with the TRIzol reagent (Invitrogen) and was reverse-transcribed with SuperScript III according to the manufacturer’s instructions. PCR analysis was performed with the primers and under the conditions described previously (34). Primers and probes from Applied Biosystems were used for the real-time PCR analysis of Cacnb1, Cacnb2, and Cacnb3 expression, and the results were quantified by analysis of the change in cycling threshold (ΔΔCt). The difference in the expression of a gene of interest (such as Cacnb2) was compared among different cell populations. We normalized the amount of input complementary DNA with the Ct value of the reference gene (Hprt). Relative gene expression was quantified with the change in cycle threshold method (ΔΔCt) as follows: (Ct of target gene expression in test sample − Ct of target gene in control sample) − (Ct of reference gene in test sample − Ct of reference gene in control sample), where control samples were always wild-type (control) T cells. All results were normalized to Hprt, which was quantified in parallel amplification reactions during each PCR.

Flow cytometry, enzyme-linked immunosorbent assay, Western blotting, and antibodies

Cells were stained with fluorochrome-conjugated antibodies in 1% (v/v) fetal bovine serum (FBS) in RPMI medium and were subsequently fixed in 2% (v/v) paraformaldehyde. The following antibodies were used for flow cytometric analysis of cells: anti-CD8α (53-6.7), anti-CD44 (IM7), anti-CD5 (53-7.3), anti-CD24 (M1/69), and anti-CD4 (RM4-5; all from BD Pharmingen). Cells were analyzed with an LSR II flow cytometer (BD Biosciences), and the data were analyzed with FlowJo software (version 6.1; Tree Star). Staining with annexin V and PI (BD Pharmingen) or CFSE (Molecular Probes) was performed according to the manufacturer’s protocol. IL-2 was detected by enzyme-linked immunosorbent assay (R&D Systems). Thymocytes were resuspended in 500 to 1000 μl of cell lysis buffer (9803; Cell Signaling) containing protease and phosphatase inhibitors, including phenylmethylsulfonyl fluoride, leupeptin, N-ethylmaleimide, and “cocktail tablets” (Roche Diagnostics), and then were briefly sonicated before being resolved by SDS–polyacrylamide gel electrophoresis and subjected to Western blotting analysis with antibodies specific for NFATc3 (R&D #AF5834), Cav1.2 (Alomone Labs #ACC-003), Cav1.3 (Alomone Labs #ACC-005), Cav2.3 (Alomone Labs #ACC-006), and actin (Santa Cruz Biotechnology).

Retroviral transduction

The gene encoding the Cre enzyme was subcloned into the mouse stem cell virus (MSCV) retroviral vector upstream of an internal ribosomal entry site–enhanced GFP (eGFP) expression cassette; thus, transduced cells could be distinguished by the coexpressed GFP. Phoenix-ECO packaging cells were transfected with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions, and recombinant retrovirus was collected 48 and 72 hours after transfection. After 24 hours of stimulation with anti-CD3 (10 μg/ml) and anti-CD28 (10 μg/ml) antibodies, total splenocytes were transduced with retrovirus by spin inoculation. The cells were then cultured for an additional 4 days before they were restimulated with anti-CD3 (10 μg/ml) for 8 hours before being analyzed by flow cytometry to assess the intracellular accumulation of IFN-γ [APC (allophycocyanin)–conjugated rat anti-mouse IFN-γ antibody, clone XMG1.2, BD Pharmingen].

Measurement of intracellular Ca2+

The concentration of intracellular free Ca2+ was measured with the ratiometric Ca2+-binding dye Fura-2 AM, as described previously (31, 34). Cells were loaded with 5 μM Fura-2 AM (Molecular Probes) for 30 min at 37°C. The cells were then stimulated with anti-CD3 antibody and anti-hamster immunoglobulin G (IgG) in a cross-linking system, and fluorescence was monitored in ratio mode (340/380 nm) with a fluorometer (POLARstar Galaxy, BMG Labtech). At the end of each experiment, the cells were treated with 5 μM ionomycin in Ca2+-containing medium. The collected data were analyzed with FLUOstar Galaxy Software (BMG Labtech).

Statistical analysis

Prism software (GraphPad) was used for statistical analyses. P values were calculated with the Student’s t test. P < 0.05 was considered to be statistically significant.

SUPPLEMENTARY MATERIALS

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Fig. S1. Cacnb2Cre/− mice have reduced numbers and proportions of peripheral T cells.

Fig. S2. DN thymocyte populations from Cacnb2Cre/− mice have an altered cell surface expression of CD5, but not CD24.

Fig. S3. Thymocytes from Cacnb2Cre/− mice exhibit defective proliferation.

Fig. S4. Cacnb2fl/fl effector T cells expressing Cre-encoding retrovirus show reduced production of IFN-γ.

Fig. S5. Cav1-deficient mice have normal percentages of thymocytes.

Fig. S6. Effect of the blockade of Cav1 channels on IL-2 production by mouse and human CD4+ T cells.

Fig. S7. Blockade of Cav1 channels impairs the expression of Nfatc3 in thymocytes.

REFERENCES AND NOTES

Acknowledgments: We thank R. LaCourse (Trudeau) for fluorescence-activated cell sorting, and C. Eaton, R. Bartiss, and H. Trumble (Trudeau) for maintaining a mouse colony at the Trudeau Institute. We thank K. Beam (Colorado) for the Cacna1smdg mutant mice, F. Hofmann (München, Germany) for the Cacna1c floxed mice, and J. Striessnig (Innsbruck, Austria) for the Cacna1d-deficient mice. Funding: We acknowledge financial support by the Deutsche Forschungsgemeinschaft SFB 894 (to P.W., V.F., and M.F.), the Howard Hughes Medical Institute (to R.A.F.), the Homburger Forschungsförderungsprogramm (to P.W.), and the Trudeau Institute (to M.K.J.). Author contributions: A.J. and A.K.S. performed the experiments and analyzed the data; P.W., M.F., V.F., and R.A.F. contributed reagents; P.W., M.F., V.F., and R.A.F. edited the manuscript; and M.K.J. conceived and designed all the experiments, directed the project, and wrote the manuscript. Competing interests: The authors declare that they have no competing interests. Data and materials availability: Cacnb2fl/fl mice were obtained under a material transfer agreement from Saarland University.
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